Aging is a process of gradual decline in cellular and bodily function. Many human diseases, such as cancers, type II diabetes, neurodegenerative diseases, are either directly or indirectly associated with aging. Therefore, aging and age-associated disease can significantly influence on the quality of daily life especially within the elderly age group. Sestrin (Sesn) is a stress-inducible gene family that can be upregulated by a variety of environmental stresses including DNA damage, oxidative stresses, hypoxia and unfolded protein stresses. Sesn has two important biological activities in reducing reactive oxygen species (ROS) and suppressing mTOR complex 1 (mTORC1), both of which may attenuate aging and its associated pathologies. Indeed, in many model animals, including worms, flies and mice, Sestrin-family proteins were shown to be a critical regulator of metabolic homeostasis that attenuates diverse age- and obesity-associated pathologies. These results suggest Sestrins to be evolutionarily conserved anti-aging molecules. However, because the biochemical basis for these anti-aging activities of Sestrins has been elusive, we were unable to harness Sestrins' beneficial activities for attenuation of aging and extension of healthspan. Based on the 3D molecular structure of human Sestrin2, which we have recently determined through X-ray crystallography, here we propose to uncover the biochemical mechanisms underlying the anti-aging activity of Sestrin proteins for the first time. In Aim 1, we will revealthe biochemical basis underlying hSesn2's antioxidant function using structure-guided mutagenesis and subsequent in vitro and in vivo assays of its redox activity. In Aim 2, using the mutant hSesn2 proteins generated from Aim 1 research, we will examine whether the mutated residues and active sites are important for suppressing mTORC1 in cultured mammalian cells and in tissues of an intact animal (Drosophila). Finally in Aim 3, dSesn-null mutant flies, which exhibit diverse accelerated aging phenotypes, will be reconstituted with wild-type and mutant hSesn2 to test if the mutated key residue(s) are functionally important for anti-aging physiological roles of Sesn. Successful completion of the proposed research will allow us to elucidate the structural basis for the physiological function of hSesn2 in suppressing aging and controlling metabolism. The structural and mechanistic information obtained from the proposed study not only reveals the mechanisms underlying Sesns' antioxidant and mTOR-inhibiting activities, but also enables development of novel small molecules that can either enhance the catalytic activity of Sesns or increase Sesn stability. These molecules, which will be developed in future, may be used to pharmacologically expand healthspan and improve the quality-of-life in the later ages.